Desacetylcephalosporin sulfones

ABSTRACT

7-(S)-Acylamino-3-hydroxymethyl-3-cephem sulfones are disclosed. These compounds are intermediates in the synthesis of 1-oxa β-lactam antibiotics.

SUMMARY OF THE INVENTION

This invention relates to cephalosporin compounds. In particular itrelates to 7-(S)-acylamino-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfones and the salts and the esters thereof, which are usefulintermediates in the preparation of 1-oxa β-lactam antibiotics.

DETAILED DESCRIPTION

This invention relates to 7-(S)-acylamino-3-hydroxymethyl cephemsulfones represented by the formula 1 ##STR1## wherein R₁ is a. C₁ to C₇alkyl, C₃ to C₇ alkenyl, chloromethyl, dichloromethyl, 4-carboxybutyl,4-formylbutyl, 4-protected carboxybutyl, 4-amino-4-carboxybutyl or4-protected amino-4-protected carboxybutyl;

b. C₁ to C₆ alkoxy, C₃ to C₆ cycloalkyloxy, benzyloxy or substitutedbenzyloxy, wherein the substituents are one to three groups chosen fromthe group consisting of C₁ to C₄ alkyl, C₁ to C₄ alkoxy and chloro;

c. 1,4-cyclohexadienyl, phenyl or substituted phenyl wherein thesubstituents are one or two groups chosen from the group consisting ofchlorine, bromine, hydroxy, trifluoromethyl, C₁ to C₄ alkyl, C₁ to C₄alkoxy, carboxy, carboxymethyl, hydroxymethyl, aminomethyl and protectedaminomethyl;

d. an arylalkyl group of the formula

    R'--(O).sub.m --CH.sub.2 --

wherein R' is 1,4-cyclohexadienyl, phenyl or substituted phenyl whereinthe substituents are one or two groups chosen from the group consistingof chlorine, bromine, hydroxy, trifluoromethyl, C₁ to C₄ alkyl, C₁ to C₄alkoxy, carboxy, carboxymethyl, hydroxymethyl, aminomethyl and protectedaminomethyl, and m is zero or one;

e. a substituted arylalkyl group of the formula ##STR2## wherein R" isR' as defined above, 2-thienyl, or 3-thienyl; W is hydroxy, carboxy orprotected carboxy, amino or protected amino;

f. a heteroarylmethyl group of the formula

    R"'--CH.sub.2 --

wherein R"' is 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 2-thiazolyl,5-tetrazolyl or 1-tetrazolyl; and

R₂ is hydrogen, a carboxylic acid protecting group, lithium cation,sodium cation, or potassium cation.

The compounds of this invention are prepared by contacting a7-(S)-acylamino-3-acetoxymethyl cephem sulfone ("3-acetoxymethylcompound") compound of the formula 2 ##STR3## in an aqueous solutionbuffered from about pH 6 to about pH 8 with the enzyme citrusacetylesterase, immobilized on silica gel. In the formula 2 R₁ is anacyl group as defined above, and R₃ is hydrogen, lithium cation,potassium cation or sodium cation.

As used in the above general description of the7-(S)-acylamino-3-hydroxymethyl cephem sulfone compounds and thecorresponding 7-(S)-acyl-3-acetoxymethyl cephem sulfone startingmaterial, the term "C₁ to C₇ alkyl" refers to methyl, ethyl, n-propyl,n-butyl, isobutyl, pentyl, n-hexyl, n-heptyl, cyclohexyl, and likealiphatic hydrocarbon chains. "C₃ to C₇ alkenyl" refers to theunsaturated hydrocarbon chains such as propenyl (allyl), butenyl,pentenyl, hexenyl, heptenyl, and the like. The term "C₁ to C₆ alkoxy"refers to methoxy, ethoxy, iso-propoxy, N-propoxy, n-butoxy, pentoxy,hexyloxy, and the like.

The term "C₃ to C₆ cycloalkyloxy" refers to cyclopropyloxy,cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.

The term "substituted benzyloxy" refers to groups such as3-chlorobenzyloxy, 2-methyl-3-chlorobenzyloxy, 2,4-dimethylbenzyloxy,4-n-propylbenzyloxy, 4-n-butylbenzyloxy, 2-ethyl-4-n-propylbenzyloxy,2-methoxybenzyloxy, 2,4-dimethoxybenzyloxy, 4-ethoxybenzyloxy,3-chloro-4-ethoxybenzyloxy, 2-methyl-3chlorobenzyloxy,4-ethoxybenzyloxy, 4-t-butylbenzyloxy, 2,4-dichlorobenzyloxy,2,3,4,-trimethoxybenzyloxy, 2,3,4-trimethylbenzyloxy,3-propoxybenzyloxy, and the like.

The term "substituted phenyl" refers to a mono- or disubstitutedhalophenyl group such as 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3-chloro-4-bromophenyl, 2-fluorophenyl, and the like; amono- or dihydroxyphenyl group such as 4-hydroxyphenyl, 3-hydroxyphenyl,2,4-dihydroxyphenyl and the like; a mono- or disubstituted loweralkylphenyl group such as 4-methylphenyl, 2,4-dimethylphenyl,2-methylphenyl, 4-isopropylphenyl, 4-ethylphenyl, 3-n-propylphenyl andthe like; a mono- or disubstituted lower alkylphenyl ether for example,2,6-dimethoxyphenyl, 4-methoxyphenyl, 3-ethoxyphenyl,4-isopropoxyphenyl, 4-t-butoxyphenyl, 3-ethoxy-4-methoxyphenyl; a mono-or disubstituted trifluoromethylphenyl group such as4-trifluoromethylphenyl, 3,4-di-(trifluoromethyl)phenyl, and the like; amono-or disubstituted carboxyphenyl group, such as 4-carboxyphenyl,2-carboxyphenyl, 3-carboxyphenyl, 2,4-dicarboxyphenyl, and the like; aphenyl ring substituted by 1 or 2 carboxymethyl groups, such as2-carboxymethylphenyl, 3-carboxymethylphenyl, 4-carboxymethylphenyl,2,3-di(carboxymethyl)phenyl, and the like; a phenyl moiety that is monoor disubstituted by hydroxymethyl, resulting in benzyl alcohol typemoieties, 2-(hydroxymethyl)phenyl, 4-(hydroxymethyl)phenyl,3-(hydroxymethyl)phenyl, 3,4-di(hydroxymethyl)phenyl, and the like;phenyl groups mono or disubstituted by amino methyl groups, resulting inbenzylamine type moieties, e.g. 2-(aminomethyl)phenyl,4-(aminomethyl)phenyl, 2,3-di(aminomethyl)phenyl, and the like. Itshould be noted that phenyl groups disubstituted with bromine areexcluded from the above definition. Also the term "substituted phenyl"also represents disubstituted phenyl groups wherein substituents can bedifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 2-hydroxy-4-chlorophenyl,3-trifluoromethyl-4-hydroxyphenyl, 2-carboxy-4-ethoxyphenyl,2-aminomethyl-4-hydroxymethylphenyl, 4-carboxymethyl-2-methylphenyl,3-hydroxymethyl-4-chlorophenyl, and like disubstituted phenyl groupsbearing different substituents.

Illustrative of the acyl groups ##STR4## when R₁ is a group oftheformula R'--(O)_(m) --CH₂ --, m is O and R' is 1,4-cyclohexadienyl,phenyl or substituted phenyl as defined above, are2-cyclohexa-1,4-dien-1-yl)acetyl, phenylacetyl,2-(4-chlorophenyl)acetyl, 2-(3-hydroxyphenyl)acetyl,2-(4-hydroxy-3-methylphenyl)-acetyl, 2-(4-hydroxyphenyl)acetyl,2-(4-bromophenyl)-acetyl, 2-(4-ethoxyphenyl)acetyl,2-(3,4-dimethoxyphenyl)acetyl, and the like; and when m is 1,representative acyl groups are phenoxyacetyl,2-(3-hydroxyphenoxy)acetyl, 2-(4-hydroxyphenoxy)acetyl,2-(4chlorophenoxy)acetyl, 2-(3,4-dichlorophenoxy)acetyl,2-(2-chlorophenoxy)acetyl, 2-(4-methoxyphenoxy)acetyl,2-(2-ethoxyphenoxy)acetyl, 2-(3,4-dimethylphenoxy)acetyl,2-(4-isopropylphenoxy)acetyl, 2-(4-methyl-2-carboxyphenoxy)acetyl,2-(4-aminomethylphenoxy)acetyl, 2-(4-carboxyphenoxy)acetyl,2-(4-carboxymethylphenoxy)acetyl, 2-(3-trifluoromethylphenoxy)acetyl,2-(hydroxymethylphenoxy)acetyl, 2-(aminophenoxy)acetyl, and like acylgroups.

Illustrative of the acyl groups ##STR5## wherein R₁ is a substitutedarylalkyl group of the formula ##STR6## wherein R" is the same as R'defined above or 2-thienyl or 3-thienyl, are the hydroxy substitutedarylalkyl groups such as the 2-hydroxy-2-phenylacetyl group of theformula ##STR7## and similar groups wherein the phenyl ring issubstituted, for example, 2-hydroxy-2-(4-methoxyphenyl)acetyl,2-hydroxy-2-(3-chloro-4-hydroxyphenyl)acetyl,2-hydroxy-2-(4-hydroxyphenyl)acetyl, 2-hydroxy-2-(3-bromophenyl) acetyl,2-hydroxy-2-(3,5-dichloro-4-hydroxyphenyl)acetyl,2-hydroxy-2-(3-chloro-4-methoxyphenyl)acetyl,2-hydroxy-2-(3-chlorophenyl)acetyl, and like groups; the2-carboxy-2-phenylacetyl group or 2-(protected carboxy)phenylacetylgroup of the formula ##STR8## and similar groups wherein the phenyl ringis substituted, for example, 2-protected carboxy-2-phenylacetyl,2-tert-butoxycarbonyl-2-phenylacetyl,2-benzyloxycarbonyl-2-(4-chlorophenyl) acetyl,2-carboxy-2-(4-hydroxyphenyl)acetyl, and like groups; and the2-amino-2-phenylacetyl or 2-(protected amino)-2-phenylacetyl group ofthe formula ##STR9## similar groups wherein the phenyl ring issubstituted, for example, 2-amino-2phenylacetyl,2-amino-2-(1,4-cyclohexadien-1-yl)acetyl,2-amino-2-(4-hydroxyphenyl)acetyl, 2-amino-2-(thien-2-yl)acetyl,2-(t-butoxycarbonylamino)-2-phenylacetyl and like acyl groups.

Representative of the acyl group ##STR10## when R₁ is a heteroarylmethylgroup of the formula

    R"'--CH.sub.2 --

wherein R"' is 2-thienyl, 3-thienylacetyl, 2-furylacetyl, 3-furylacetyl,a 2-thiazolylacetyl group of the formula ##STR11## a2-(1-tetrazolyl)acetyl group of the formula ##STR12## or a2-(5-tetrazolyl)acetyl group of the formula ##STR13##

The carboxy and amino groups present in the starting materials of theprocess for preparing the compounds of this invention are optionallyprotected; however, the 4-carboxylic acid group of the starting materialmust be in a soluble salt form for the process for preparing thecompounds of this invention. It may be convenient to protect othercarboxy or amino groups in the starting material before the process forpreparing the compounds of this invention is carried out, since in thesubsequent steps required to convert the product compounds to theultimate products, the 1-oxa β-lactam antibiotics, protection of one ormore of such groups is required. Suitable amino and carboxy protectinggroups for the compounds of this invention will be discussed below.

Examples of the 7-(S)-acylamino-3-hydroxymethyl-3-cephem-4carboxylicacid sulfone compounds of this invention include:

Benzyl7-(S)-[2-(fur-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Phenacyl7-(S)-[2-(tetrazol-1-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Dimethyallyl7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

p-Chlorophenylacyl7-(S)-[2-hydroxy-2-(3-chlorophenyl)acetamido]-3-cephem-4-carboxylatesulfone,

Dimethylallyl7-(S)-[2-hydroxy-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Phenacyl7-(S)-(2-amino-2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzyl7-(S)-[2-amino-2-(4-hydroxyphenyl)-acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Dimethylallyl7-(S)-(2-aminomethylphenyl)acetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-(fur-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[2-(tetrazol-1-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Lithium7-(S)-[2-hydroxy-2-(3-chlorophenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[2-hydroxy-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Lithium7-(S)-(2-amino-2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-(2-aminomethylphenyl)acetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

7-(S)-[2-(fur-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-[2-(tetrazol-1-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylicacid,

7-(S)-[2-hydroxy-2-(3-chlorophenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-hydroxy-2-(4-hydroxyphenyl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-(2-amino-2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-(2-aminomethylphenyl)acetamido-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone.

The preferred 7-(S)-acylamino-3-hydroxymethyl-3 -cephem-4-carboxylicacid sulfones of this invention include:

Potassium 7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxlate sulfone,

Potassium 7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium 7-(S)-phenoxyacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium 7-(S)-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

Potassium7-(S)-[D-(5-(2,4-dichlorobenzyloxy-carbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

4-Methoxybenzyl7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-((4-methoxybenzyl)carboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl 7-(S)-[D-(5-benzyloxycarbonylamino)-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

4-Methoxybenzyl7-(S)-[D-(5-(4-methoxybenzyloxycarbonylamino))-5-((4-methoxybenzyl)carboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-((t-butyl)carboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

t-Butyl7-(S)-[D-(5-(4-methoxybenzyloxycarbonylamino))-5-((t-butyl)carboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

t-Butyl 7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

4-Methoxybenzyl7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

t-Butyl 7-(S)-phenoxyacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

4-Methoxybenzyl7-(S)-phenoxyacetamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

4-Methoxybenzyl7-(S)-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

7-(S)-Benzamido-3-hydroxymethyl-3-cephem-4-carboxylic acid sulfone,

7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-Phenoxyacetamido-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-Phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-Benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone, and

7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone.

The more preferred 7-(S)-acylamino-3-hydroxymethyl-3-cephem-4-carboxylicacid sulfone compounds of this invention include:

Benzhydryl 7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl 7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl 7-(S)-phenoxyacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl 7-(S)-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Benzhydryl7-(S)-benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

Benzhydryl7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

Sodium 7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

Sodium 7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-phenoxyacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

Sodium 7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(sodiumcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone.

In general, the process for preparing the7-(S)-acylamino-3-hydroxymethyl-3-cephem sulfones (formula 1) involvesdeacetylating a 3-acetoxymethyl compound (formula 2) with citrusacetylesterase immobilized on a functionalized silica gel. The processis carried out by contacting an aqueous solution of the 3-acetoxymethylcompound buffered at a pH of between about 6 and about 8 with citrusacetylesterase immobilized on the functionalized silica gel.

The term "functionalized silica gel" as used herein refers to anaminosilanized silica gel functionalized with an alkane dialdehyde. Theacetylesterase enzyme is covalently bonded to the functionalized silicaand thus is insolubilized or immobilized thereon and accessible to thebuffered solution of the acetoxymethyl compound.

In carrying out the process, the buffered solution of the3-acetoxymethyl compound can be passed through a column packed with theimmobilized enzyme, or alternatively, the immobilized enzyme and thebuffered solution can be mixed in a suitable vessel such as a tank orglass flask. Preferably, the solution is passed through a column loadedwith the immobilized acetylesterase enzyme.

As stated above, the starting materials for the instant process aredissolved in an aqueous solution of buffer. The amount of startingmaterial dissolved in the aqueous buffer solution is limited only by thesolubility of the starting material. The usual range of concentration ofthe starting material is between about 1 to about 3% w/v. The typicalvalue in this range is 2%, a value obtained with 7-(S)-(2phenoxyacetamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone.

The buffering agents used in the instant process are not critical solong as they are used in such a concentration as to keep the aqueoussolution containing the starting materials and/or products at a pHbetween about 6 and about 8. Buffers that can be used are those known inart, e.g., sodium citrate, sodium phosphate, a combination of potassiumdihydrogen phosphate and disodium hydrogen phosphate,tris(hydroxymethyl)aminomethane, 4-morpholinepropane sulfonic acid(MOPS), piperazine-N,N'-bis(2-ethane sulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid (TES),N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid (HEPES), and thelike. General methods for employing the potassium dihydrogenphosphate/disodium hydrogen phosphate buffer and thetris(hydroxymethyl)aminomethane buffers are found on pages D-186 andD-187 of the Handbook of Chemistry and Physics, 59th edition, CRC Press,West Palm Beach, Fla., 33409.

General procedures for using the MOPS, PIPES, TES, and the HEPESbuffering agents are found in N. E. Good et al., Biochemistry, 5, 1966,page 467.

It will be appreciated that as the concentration of the startingmaterial is increased in the aqueous buffered solution, it is necessaryto increase the amount of buffering agent used in order to neutralizethe increased amount of acetic acid that is produced in the instantdeacetylation procedure. The preferred buffering agents for thisprocedure are the sodium citrate and sodium phosphate buffers, e.g.,0.2N sodium citrate, 0.3M sodium phosphate, and a mixture of 0.05Msodium citrate and 0.075M sodium phosphate, and 0.2M sodium phosphate.

As stated above, it is necessary to use a sufficient amount of bufferingagent in the aqueous solution containing the starting material for theinstant process to maintain a pH between about 6 to about 8. Thepreferred pH range is between about 6 to about 6.5.

The citrus acetylesterase enzyme is known in the art. Specifically, thecharacteristics and suggested isolation procedure for the enzyme arefound in Methods in Enzymology, 43, p. 728, 1975, Academic Press, N.Y.The procedure for isolating the enzyme in the instant process is derivedfrom the procedure described in the above reference.

Suggested particle size for the silica gel used as a support for theimmobilized enzyme in the instant process is between about 0.06 mm toabout 0.20 mm although this is not a critical parameter.

The method for immobilizing the citrus acetylesterase enzyme in theinstant process involves:

(1) Derivatizing the silica gel with an amino organosilane. Suitableamino organosilanes include for example, aryl amino and alkylaminosilanes such as aminophenyltriethoxysilane, and3-aminopropyltriethoxysilane. The preferred amino organo silane is3-aminopropyltriethoxysilane (also calledtriethoxysil-1-yl-3-amino-propane).

(2) Activating the derivatized support. The above aminosilanized silicagel is reacted with an alkane dialdehyde crosslinking agent in order tofacilitate bonding to the enzyme. Alkane dialdehyde cross-linking agentswhich can be reacted with a derivatized silica gel include C₂ to C₁₀alkane dialdehydes such as glyoxal, malonaldehyde, succinaldehyde,glutaraldehyde, adipaldehyde, pimelicdialdehyde and subericdialdehyde.The preferred alkane dialdehyde crosslinking agent used to activate thederivatized support is glutaraldehyde.

(3) Immobilizing the enzyme. The citrus acetylesterase is then reactedwith the above activated derivatized support.

The flow rate of starting material-containing buffered solution throughthe immobilized enzyme is set so that at least 90% of the startingmaterial, 7-(S)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone, is converted to the corresponding 3-hydroxymethyl compound. Theappropriate flow rate is determined by percolating a small amount of thebuffered solution through a column packed with immobilized enzyme andmonitoring the conversion by high performance liquid chromatography. Theflow rate is adjusted in order to achieve a conversion of 90% orgreater. Once the flow rate has been determined the reaction isperiodically monitored by high performance liquid chromatography inorder to maintain the appropriate flow rate necessary to achieve thehighest percent conversion. Periodic monitoring is preferred because thecitrus acetylesterase activity will gradually decrease over a period oftime, thus necessitating a decrease in the flow rate in order toincrease the residence (contact) time of the starting material on thecolumn. Typical flow rates include 30 ml/hr to 400 ml/hour.

The process is carried out at a temperature of between about 0° C. toabout 30° C. The preferred temperature is ambient room temperatures.

The product 7-(S)-acylamino-3-hydroxymethyl-3-cephem-4-carboxylic acidsulfone is isolated from the effluent stream of the immobilized esterasereactor by acidifying the effluent from the column with e.g. 1Nhydrochloric acid, and then extracting the free carboxylic acid productinto an organic solvent such as ethyl acetate. The deacylated sulfonemay also be recovered as the carboxylic acid salt by adding sodium2-ethylhexanoate to the combined ethyl acetate extracts or alternativelychanging solvents from ethyl acetate to methanol and then adding sodiumacetate.

The general procedure for the preparation of the citrus acetylesteraseenzyme, its immobilization, and the use of the immobilizedacetylesterase to deacetylate7-(S)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfones isas follows:

(1) Silica gel derivatization

Silica gel (E. Merck and Co., Fractosil 200, 70-230 mesh, 62-200μparticle size) is cleaned by deaerating under vacuum a slurry in aqueous10% nitric acid, heating the slurry for 3 hours at 80° C., then rinsingwith water. This clean silica gel is slurried in 5-10 v/w 10%3-aminopropyltriethoxysilane, then deaerated by vacuum. The pH of theslurry is adjusted to 3-4 with dilute hydrochloric acid, and the slurryis then agitated occasionally during 3 hours of heating at approximately80° C. The derivatized silica gel is collected on a suction filter,washed with 1 volume of water, and then is dried for about 16 hours at105° C.

(2) Support Activation

The aminosilanized silica is slurried in 5-10 v/w of a 3% aqueousglutaraldehyde, buffered at pH 7 with a phosphate buffering agent. Theslurry is agitated occasionally during a 3 hour period. The resultantactivated derivatized silica gel is then washed with water and citratebuffer at a pH of 7.

(3) Enzyme immobilization

A neutral aqueous solution of citrus acetylesterase enzyme is added tothe activated derivatized silica gel and this mixture is allowed tointeract for between about 4 to about 20 hours. The immobilized enzymeis transferred to a glass or metal column and is washed by percolationof sodium citrate buffer at pH 7 through the packed bed. A solution ofthe 3-acetoxymethyl-3-cephem cephalosporin sulfone acid in sodiumcitrate buffer at pH 7 (1-3% concentration) is percolated through theimmobilized enzyme. The effluent from the column is monitored by highpressure liquid chromatography, and the flow rate is adjusted to allow aresidence time sufficient to provide a 90+% deacetylation of the3-acetoxymethyl sulfone.

Examples of starting materials that can be used for the instant processinclude:

7-(S)-[2-(fur-2-yl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-[2-(tetrazol-1-yl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-hydroxy-2-(3-chlorophenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-hydroxy-2-(4-hydroxyphenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-(2-amino-2-phenylacetamido)-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-(2-aminomethylphenyl)acetamido-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

Sodium7-(S)-[2-(fur-2-yl)acetamido]-3acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[2-(tetrazol-1-yl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Lithium7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-hydroxymethyl-2-(3-chlorophenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[2-hydroxy-2-(4-hydroxyphenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Lithium7-(S)-(2-amino-2-phenylacetamido)-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-(2-aminomethylphenyl)acetamido-3-acetoxymethyl-3-cephem-4-carboxylatesulfone.

Preferred starting materials for the instant process include:

Potassium 7-(S)-benzamido-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-(para-toluylamido)-3-acetoxymethyl-3-cephem-4-carboxylate sulfone,

Potassium 7-(S)-phenoxyacetamido-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium 7-(S)-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone,

Potassium7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylate sulfone,

Sodium7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-((4-methoxybenzyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[D-(5-(4-methoxybenzyloxycarbonylamino))-5-((4-methoxybenzyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-((t-butyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Potassium7-(S)-[D-(5-(4-methoxybenzyloxycarbonylamino))-5-((t-butyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-((4-methoxybenzyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[D-(5-(4-methoxybenzyloxycarbonylamino))-5-((4-methoxybenzyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-((t-butyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-[D-(5-(4-methoxybenzyloxycarbonylamino))-5-((t-butyl)carboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone.

More preferred substrates for the instant process include:

7-(S)-Benzamido-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone,

7-(S)-(p-toluylamido)-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-Phenoxyacetamido-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-Phenylacetamido-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-[2-(thien-2-yl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid sulfone,

7-(S)-Benzyloxycarbamido-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(benzyhydrylcarboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

Sodium 7-(S)-benzamido-3-acetoxymethyl-3-cephem-4-carboxylate sulfone,

Sodium 7-(S)-(p-toluylamido)-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-phenoxyacetamido-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone,

Sodium 7-(S)-phenylacetamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone,

Sodium7-(S)-[2-(thien-2-yl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-benzyloxycarbamido-3-acetoxymethyl-3-cephem-4-carboxylatesulfone,

Sodium 7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(sodiumcarboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylate sulfone,

Sodium 7-(S)-[D-(5-(2,4-dichlorobenzyloxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylate sulfone.

The starting materials employed in the process for making the compoundsof this invention are obtained by oxidizing a7-(R)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid to thecorresponding sulfone. The7-(R)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone isthen epimerized to the corresponding 7-(S)-acylamino compound.

The above sequence of reactions is illustrated by the following reactionscheme: ##STR14##

In the above formulas R₁ is as defined for formula 1.

The preparation of the7-(R)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone isbest carried out in an aqueous reaction medium maintained at a pHbetween about 5.0 and about 6.0 with an excess of potassium hydrogenpersulfate. The oxidation proceeds well at temperatures of about 15° toabout 45°. The sulfone is recovered from the aqueous reaction medium byacidifying the mixture to form the free sulfone carboxylic acid andextraction of the latter with a suitable water immiscible solvent suchas ethyl acetate.

The epimerization of the7-(R)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone ispreferably carried out in an aqueous medium as follows. A slurry of thesulfone free acid in water is treated with an aqueous solution of sodiumacetate containing at least an equimolar amount of sodium acetate. Anaqueous solution of piperazine is then added dropwise until the pH ofthe solution is about 9.5 to 10. With the pH adjusted, the epimerizationmixture is stirred for about 5 to 15 minutes and the product recoveredas follows. Ethyl acetate is added to the mixture which is thenacidified to a pH of about 2.0 with concentrated hydrochloric acid. The7-(S)-acylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone isthen extracted with ethyl acetate.

The preparation of the cephalosporin sulfones and the epimerization tothese 7-(S)-acylamino sulfones are described in co-pending applicationSer. Nos. 442,079 and 442,077 filed this even date.

The 7-(S)-acylamino-3-hydroxymethyl 4-carboxylic acid sulfone compoundsof this invention as discussed above are intermediates in the synthesisof 1-oxa β-lactam antibiotics.

The first step of this synthesis of 1-oxa β-lactam involves electrolyticreduction of the 3-hydroxymethyl sulfone compounds of this invention to2-(R)-sulfinic acid-3-(S)-acylamino azetidinones, represented by formula3 ##STR15## wherein R₁ is as described for formula 1, M is a lithium,potassium, sodium, ammonium or a substituted ammonium cation, and R ishydrogen, a carboxylic acid protecting group, lithium, potassium,sodium, ammonium or substituted ammonium cation. The sulfinic acidazetidinones of formula 3 are prepared as described in co-pendingapplication Ser. No. 442,075, filed this even date. As describedtherein, the 7-(S)-acylamino-3-hydroxymethyl-4-carboxylate sulfonecompounds of the instant invention are reduced at the cathode of anelectrolysis cell usually comprised of a mercury pool cathode, aplatinum anode, and a cationic resin separating the cathode and anodecompartments. A suitable electrolyte can be sodium perchlorate, lithiumperchlorate or sodium acetate. The anolyte is preferably a phosphatebuffer at pH 2.7.

The reduction is carried out preferably at a temperature between about-10° C. and about 10° C. and at a reduction potential between about -1.0V and about -1.9 V vs. a standard calomel electrode.

The 3-hydroxymethyl sulfone ester is dissolved in methyl alcoholcontaining a proton source such as an organic acid, e.g. acetic acid.The electrolyte of choice, e.g. sodium acetate, is then added to thesolution to achieve a 0.1M concentration of the electrolyte.

The solution is then placed in the cathode compartment and theelectrolysis apparatus is flushed with argon until any oxygen present isremoved. The electrolysis can be carried out at constant potential or atconstant current. The progress of the electrolysis can be followed byuse of analytical HPLC on an aliquot of the reduction mixture.

Following the electrolysis the reduction product mixture is extracted inthe cold with a water immiscible organic solvent, preferably ethylacetate.

The above electrolysis procedure to produce the sulfinic acidazetidinones of formula 3 produces 2 isomers of this general class ofsulfinic acids, i.e. the β,γ azetidinone sulfinic acid compoundsrepresented by formula 4 ##STR16## or the α,β azetidinone sulfinic acidcompounds represented by formula 5 ##STR17##

The carboxy groups of the 7-(S)-acylamino-3-hydroxymethyl-3-cephemsulfones (formula 1) should be protected during the electrolysis when anorganic solvent or an organic solvent plus water is used.

Accordingly, the choice of carboxy protecting groups should be limitedto ones that are stable to the electrolysis process (i.e., not easilyreduced), thus ruling out protecting groups such as p-nitrobenzyl,2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2-di-bromoethyl,2-iodoethyl, 2,2-diiodoethyl, 2,2,2-tri-iodomethyl, and like protectinggroups having nitro, activated halogeno or cyano substituents. Inaddition, the carboxy protecting groups used must not be so acid-labileas to be removed by the proton source used in the electrolysis, i.e. thecarboxylic acid having a pKa between about 0 to about 5. Acid-labileprotecting groups that should be avoided are the silyl groups such astrimethylsilyl. If these limitations are heeded, protecting groups thatcan be used on either the starting materials of formula 1 or theproducts of formulas 4 and 5 of the electrolysis process where the term"protected carboxy" is specified are those commonly used carboxylic acidprotecting groups such as tert-butyl, benzyl,diphenylmethyl(benzhydryl), 4-methoxybenzyl, C₂ -C₆ alkanoyloxymethyl,phenacyl, chlorophenacyl, dimethylallyl, and the like. Preferredcarboxylic acid protecting groups are benzhydryl, 4-methoxybenzyl andtert-butyl.

It is not essential that amino groups present in the3-hydroxymethyl-3-cephem sulfones be protected in the electrolysisprocess. However, if unprotected, it is necessary to take their presenceinto account when calculating the amount of proton source needed in theelectrolytic reduction to an azetidinone sulfinic acid (formula 3), asthe unprotected amino groups will consume some of the protonconcentration needed for the electrolysis reduction. If anamino-protecting group is used, it is again necessary, as withcarboxylic acid protecting groups, to avoid the use of easily reduciblegroups, e.g. the 4-nitrobenzoxycarbonyl group, the2,2,2-trichloroethoxycarbonyl, the β-haloethyloxycarbonyls, etc. Withthese precautions observed, the protecting groups that can be used insituations where "protected amino" groups are specified are those knownin the cephalosporin art, such as the benzyloxycarbonyl group, the2,4-dichlorobenzyloxycarbonyl group, and the 4-methoxybenzyloxycarbonylgroup.

In the foregoing definitions, amino and carboxy protecting groups arenot exhaustively defined. Many such protecting groups are well known inthe art and the use of other groups equally applicable to the compoundof this invention, such as those described in Theodora W. Greene,"Protective Groups In Organic Synthesis", John Wiley and Sons, 1981,N.Y. will be recognized as suitable. Thus, there is no novelty orinventiveness asserted with regard to the "protecting groups" in thisspecification.

The β,γ-azetidinone sulfinic acid compounds produced by the electrolysisprocedure are converted to a β,γ epi-oxazoline compound of the formula 6##STR18##

The conversion to the β,γ epi-oxazolines is carried out by reacting theazetidinone sulfinic acid compounds with an oxidizing agent. Thereaction generally is carried out by mixing at least one molarequivalent and up to about 1.5 molar equivalents of the oxidizing agentwith each molar equivalent of the β,γ-azetidinone sulfinic acidcompound. Preferably, the ratio of reactants is from about 1.0 to about1.1 molar equivalents of oxidizing agent per molar equivalent of theazetidinone sulfinic acid compound. Preferably, the reaction is carriedout in a suitable inert organic solvent at a temperature from about 0°C. to about 30° C., for a period sufficient for the completion of thereaction. This oxidation reaction can be carried out on an azetidinonesulfinic acid compound that has been isolated and/or purified, or theoxidizing agent can be added directly to the catholyte of the precedingelectrolysis after it has been removed from the cathode compartment.

The term "inert organic solvent" means an organic solvent which, underthe conditions of the epi-oxazoline formation, does not appreciablyreact either with the reactants or with the products. Suitable inertorganic solvents include, for example, aromatic hydrocarbons, such asbenzene, toluene, ethylbenzene, cumene, and the like; halogenatedhydrocarbons, such as carbon tetrachloride, chlorobenzene, bromoform,bromobenzene, methylene chloride, ethylene chloride,1,1,2-trichloroethane, ethylene dibromide, and the like; amides, such asN,N-dimethylformamide, and the like; alcohols, such as methanol,ethanol, and the like; esters, such as ethyl acetate, and the like;nitriles, such as acetonitrile, and the like; and the other appropriateinert solvents. Preferred solvents include N,N-dimethylformamide,acetonitrile, ethylacetate, methylene chloride, and the like.

The oxidizing agent used in this reaction can be any of a wide range ofsuch agents. Typical agents include, for example, lead (IV) compoundssuch as lead tetraacetate, lead oxide, and the like; manganese (IV)compounds, such as manganese acetoacetonate, manganese oxide, and thelike; sodium hypochlorite; N-haloimides, such as N-bromosuccinimide, andthe like; ammonium cerium nitrate; and other like oxidizing compounds.Preferably, the oxidizing agent is a lead (IV) compound, in particular,lead tetraacetate, or an N-bromoimide, in particular,N-bromosuccinimide.

The temperature of the oxidation reaction generally is from about 0° C.to about 30° C. Preferably, the reaction temperature is at the lower endof this range, generally from about 0° C. to about 5° C.

Typically the oxidation reaction is complete in a very short time,generally a matter of a few minutes on small scale reactions. Normallythe time of the reaction will be no longer than about 1 hour.

The above oxidation reaction is more fully described and claimed in U.S.application Ser. No. 442,052, filed this even date.

The β,γ-epi-oxazoline compounds discussed above are converted to the1-oxa β-lactam compounds as described in U.S. Pat. Nos. 4,220,766,4,271,295 and 4,271,296 herein incorporated by reference.

The β,γazetidinone sulfinic acid compounds wherein the C-3 substituentis a carbamato side chain, i e., an azetidinone sulfinic acid compoundof the formula 7 ##STR19## wherein R₆ is C₁ to C₆ alkoxy, C₃ to C₆cycloalkyloxy, benzyloxy or substituted benzyloxy as described above areproduced by the electrolysis process discussed above. The aboveβ,γ-azetidinone sulfinic acid compounds of formula 7 are converteddirectly to an isomeric mixture of a cyclization product, i.e., a3-exomethylene 1-oxa β-lactam compound of the formula ##STR20## and a3-methyl 1-oxa β-lactam compound of the formula

The azetidinone sulfinic acid (formula 7) is reacted in an inert organicsolvent with lead tetraacetate in liquid sulfur dioxide in the presenceof copper (II) ion at a temperature between about -25° C. and about 0°C. Between about 1.0 and about 2.5 molar equivalents of leadtetraacetate per molar equivalent of azetidinone sulfinic are used.

The amount of sulfur dioxide used can be between about 1 to about 3molar equivalents per molar equivalent of substrate azetidinonecompound, preferably in excess of the molar equivalents of substratecompound is used. The sulfur dioxide can be used as a solvent by itselfor in addition to an inert organic solvent such as ethyl acetate,methylene chloride, tetrahydrofuran, dioxane, and the like.

A readily available source of copper (II) ion is copper sulfate. Betweenabout 10 to about 15 milligrams of copper sulfate per millimole ofazetidinone sulfinic acid compound is a suitable amount.

The 3-exomethylene 1-oxa β-lactam compound obtained can be easilyisomerized to the 3-methyl 1-oxa β-lactam compound in the presence of abase such as triethylamine. The 3-methyl 1-oxa β-lactam compounds areintermediates in the synthesis of 1-oxa β-lactam antibiotic compounds,such as those described in U.S Pat. Nos. 4,226,866 and 4,138,486.

The cyclization reaction of the 3-carbamato azetidinone sulfinic acidcompound (formula 7) to the 1-oxa β-lactam intermediates is described inco-pending U.S. application, Ser. No. 442,080, filed this even date. Theconversion of the 1-oxa β-lactam intermediate produced in thiscyclization process to the 1-oxa β-lactam antibiotic compounds isdescribed in the above co-pending U.S. application and U.S. Pat. Nos.4,226,866 and 4,138,486, said patents here incorporated by reference.

The α,β-azetidinone sulfinic acid compounds of the above formula 5provided by the electrolysis of a formula 1 compound encompass both the2'-Z isomer, represented by the partial formula ##STR21## and the 2'-Eisomer, represented by the partial formula ##STR22##

In the formula 5, R₅ is 1,4 cyclohexadienyl, phenyl or substitutedphenyl as described for a compound of formula 1.

The above α,β sulfinic acids (formula 5) are minor products in theelectrolytic process for making the β,γ sulfinic acid compounds. Theisomerization of the double bond from the β,γ positions to the α,βposition is caused by basic species in the catholyte and/or the work-upprocedure. In this regard, the cathode salt used is especially effectivein catalyzing the isomerization of the double bond, and in particularthe tri-n-butylammonium para-toluylsulfonate salt has been found to givethe largest amount of the α,β sulfinic acid isomer. A specific set ofreaction conditions for the electrolysis process that will produce theα,β sulfinic acid isomer exclusively involves the use of methanol as theliquid medium, acetic acid as the proton source, tri-n-butylammoniumpara-toluylsulfonate as the catholyte salt with the temperature of thereaction between about -10° C. to about 10° C.

The α,β azetidine sulfinic acids discussed above are converted to thecorresponding α,β-unsaturated epi-oxazoline of the formula ##STR23##prepared by the same process used for the β,γ sulfinic acid compounds.In the above formula for the α,β epi-oxazoline compounds R₅ is the sameas for the corresponding α,β sulfinic acids, as discussed above. Theseα,β epi-oxazoline compounds are then converted to 1-oxa β-lactamantibiotic compounds using the same processes as for the β,γepi-oxazoline compounds, as discussed above.

The following examples and preparations are supplied to furtherillustrate the instant invention, and are not meant to limit the scopeof the invention. As used in the following examples, the abbreviations"IMER", "n.m.r." and "F.A.B.M.S." stand for immobilized enzyme reactor,nuclear magnetic resonance and fast atom bombardment mass spectrum,respectively. Unless otherwise noted, all n.m.r. spectra were taken inDMSO-d₆ at 90 MHZ. DMSO, assigned a value of δ2.49, is used as thereference in the n.m.r. spectra, unless otherwise noted. Alltemperatures are in degrees centigrade.

PREPARATION 1 Preparation of porous silica gel carrier

a. Amino-silanization.

Silica gel (FRACTOSIL® 200 particle size 120-230 mesh, 63-125 mμ, meanpore diameter 9 nm, E. Merck Co., P.O. Box 2000, Rahway, N.J. 07065, 100g) was cleaned by addition to 10% nitric acid (100 ml). The slurry wasevaporated in vacuo, then heated on a steam bath (2 hours) withoccasional stirring. The porous silica gel was collected by suctionfiltration and washed with copious amounts of deionized water. The cleanporous silica gel was added to a 10% aqueous solution oftriethoxysil-1-yl-3-aminopropane (100 ml, liquid reagent obtained fromAldrich Chemical Company) and the pH of the resultant mixture wasadjusted to about 3.5 to about 4.0 by the addition of 4N hydrochloricacid. The mixture was heated on a steam bath (approximately 2 hours)with occasional stirring. The amino-silanized porous silica gel wascollected by suction filtration, washed with deionized water (250 ml),then dried in an oven (110°) overnight.

b. Condensation of amino-silylized porous silica gel with glutaraldehyde

(1) Amino-silanated FRACTOSIL® 200 (25 g, from a. above) was added to amixture of 8% aqueous glutaraldehyde solution (100 ml) and 0.3M pH 7phosphate buffer (150 ml). The mixture was dearated under vacuum, andthen swirled occasionally over a 3 hour period. The condensedamino-silanated porous silica gel carrier was collected by suctionfiltration and washed with copious amounts of 0.3M pH 7 phosphatebuffer.

(2) The above procedure (b. 1) is carried out, substituting 0.2M sodiumcitrate buffer (pH 7, 300 ml) for the phosphate buffer, doubling theamount of glutaraldehyde solution used, and using more amino-silanatedFRACTOSIL® 200 (80 g vs. 25 g) than is used in b. 1.

PREPARATION 2 Preparation of the immobilized enzyme reactor (IMER)

a. Enzyme preparation

Fresh orange peel (2000 g) was blended in 500 g batches with 2 volumesof cold 0.25M sodium chloride solution for 1.5 minutes each in a largeWaring blender. The resultant slurry was stirred in a chillroom for 1hour, while maintaining the pH between 7.0 to 7.5 with 10% sodiumhydroxide solution. The resultant solids were filtered on four layers ofcheesecloth by suction filtration, and the filtrate was lyophilized toyield 250 g of solids. A portion of these lyophilized solids (192 g) wassuspended in cold water (960 ml, total volume), then stirred for 10minutes. The insoluble material was removed from this solution bycentrifugation and filtration through filter paper. Decolorizingcharcoal (3% w/v) was added to the filtrate and stirred for 10 minutes.The charcoal was then removed from the filtrate by centrifugation andfiltration through packed glass wool. To the resultant filtrate (1200ml) was added ammonium sulfate (197 g), and the solution was stirred for1 hour. The insoluble material was removed by centrifugation anddiscarded. To the cold centrifugate solution was added an additionalportion of ammonium sulfate (235 g) to provide 60% saturation, and thissolution was stirred for 2 hours and the resultant solids were separatedby centrifugation. The solids were dissolved in 0.002M potassiumhydrogen phosphate buffer (150 ml), then dialyzed overnight againstbuffer (5 L). The solution was then centrifuged to remove a slightprecipitate which formed, yielding 236 ml of the enzyme preparation.

b. Column preparation

The enzyme solution from a. above was added to the activated porousamino-silanized silica gel from Preparation 1 b. 1. The mixture wasswirled occasionally at room temperature for about 2 hours, thenrefrigerated. The porous silica gel, now containing the immobilizedacetyl-estearase, was packed into a 60 ml cylindrical dropping funneland the column was washed with 0.3M phosphate buffer (pH 7), then 0.1Msodium citrate buffer (pH 7.0).

EXAMPLE 1 Sodium 7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone

7-(S)-Benzamido-3-acetoxymethyl-3-cephem-4-carboxylic acid sulfone (13.2g) was dissolved in 0.2M aqueous sodium citrate solution (38.9 g sodiumcitrate in 630 ml water). The pH of this solution was adjusted to 7.0 bythe addition of 1.0 N sodium hydroxide solution (approximately 30 ml).The solution was then percolated through the IMER (40-50 ml/h). Theeffluent (680 ml) collected from the IMER was layered with ethyl acetate(800 ml), chilled to 0° C. in an ice-alcohol bath, then the pH wasadjusted to 2.5 by the addition of 1 N hydrochloric acid. The layerswere separated and the aqueous layer was extracted with ethyl acetate(400 ml, 2×). The ethyl acetate extracts and the ethyl acetate layerwere combined. Ice was added to the combined ethyl acetate solutionwhich was then washed with an acidified saturated aqueous sodiumchloride solution (250 ml, 2×). The solution was dried over magnesiumsulfate and filtered. Sodium acetate (2.8 g) was added to the filtrateand the solution was stirred at room temperature for 3 h. andrefrigerated overnight. The cream-colored crystals which deposited werecollected by suction filtration, then washed with cold ethyl acetate.The crystals were allowed to air-dry first and then were dried in vacuoto yield 8.6 g of sodium7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxylate: F.A.B.M.S.(m+1)=389.

EXAMPLE 2 Benzhydryl7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone

The IMER from Example 1 was washed with 0.2 M aqueous sodium citratesolution (approx. 250 ml). Additional eluant and IMER washingscontaining the equivalent of 14 g of starting material were layered withethyl acetate (1 L), then chilled to 0° C. in an ice-alcohol bath.Diphenyldiazomethane (5.0 g) was added to the stirred, chilled solution,and the pH of the resultant solution was adjusted to 2.4 by the additionof 1 N hydrochloric acid. After 3 h, the phases were separated, and theaqueous layer was extracted with ethyl acetate (400 ml, 2×). The ethylacetate extracts and the ethyl acetate layer were then combined. Theethyl acetate solution was washed with 0.3 M phosphate solution (pH 7,400 ml, 2×) and with saturated aqueous sodium chloride (1×), dried overmagnesium sulfate, filtered, and the solvent removed in vacuo to yieldthe benzhydryl ester as 11.4 g of a yellow foam. The foam was trituratedwith diethyl ether (approx. 75 ml) resulting in the formation of a gum.The mother liquor was decanted and the gum was dissolved in ethylacetate (400 ml). The ethyl acetate solution was then washedsequentially with acidified saturated sodium chloride (250 ml, 2×), 0.3M phosphate solution (250 ml, 2×, pH 7) and saturated sodium chloridesolution (1×). The ethyl acetate solution was then dried over magnesiumsulfate, filtered, and the solvent was removed to yield 10 g ofbenzhydryl 7-(S)-benzamido-3-hydroxymethyl-3-cephem-4-carboxylatesulfone: n.m.r. δ 4.08 (s, 2, 3'-CH₂), 4.26 (dd, 2, C₂ -H), 5.23 (dd, 1,C₇ -H), 5.62 (d, 1, C₆ -H), 6.94 (s, 1, benzhydryl methine), proton7.2-8.0 (m, 15, aromatic protons), 9.54 (d, 1, amido proton).

EXAMPLE 3 Benzhydryl7-(S)-(p-toluylamido)-3-hydroxy-methyl-3-cephem-4-carboxylate sulfone

A 1% solution of7-(S)-(p-toluylamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid in 0.3M phosphate buffer (pH 6.8) was percolated through the IMER prepared asdescribed above. The effluant from the IMER column (400 ml) was layeredwith ethyl acetate (700 ml) and chilled to -1° C. in an alcohol-icebath. Diphenyldiazomethane (1.3 g) was added to the chilled solution andthe pH of the solution was adjusted to 2.5 by the addition of 1 Nsulfuric acid. The layers were separated after 0.75 h, and the aqueouslayer was extracted with ethyl acetate (75 ml) and the extract wascombined with the organic layer. After approximately 1.5 hours, theorganic layer was red-orange. The organic layer was washed further withaqueous phosphate solution (pH 7.0, 2×), and with saturated aqueoussodium chloride solution (1×), dried over magnesium sulfate, filteredand the solvent removed by evaporation to yield 2.97 g of benzhydryl7-(S)-(p-toluylamido)-3-hydroxymethyl-3-cephem-4-carboxylate sulfone:n.m.r. (100 MHz) δ 2.47 (s, 3, methyl of toluylamido), 4.11 (d, 2, C₃'-H), 4.29 (ABq, 2, C₂ -H), 5.26 (dd, 1 C₇ -H), 5.64 (d, 1, C₆ -H), 6.96(s, 1, benzhydryl methine), 7.2 to 7.8 (m, 14, aromatic protons) 9.48(d, 1, amido proton).

EXAMPLE 4 Sodium7-(S)-(2-phenoxyacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone

7-(S)-(2-Phenoxyacetamido)-3-acetoxymethyl-3-cephem-4-carboxylic acidsulfone was dissolved in a 0.2 M sodium citrate solution (pH approx.7.2, adjusted with 1 N sodium hydroxide) until the solution wasapproximately 2% in the sulfone starting material. The sulfone solutionwas filtered and percolated through the IMER (200 ml/h, later at 100ml/h) until a total of 90 g of the sulfone had been treated on the IMER.

IMER effluent (1000 ml) was layered with ethyl acetate (1000 ml) andthis mixture was chilled to 0° C. in an ice-alcohol bath. The pH of thechilled solution was adjusted to 2.5 by the addition of 1 N hydrochloricacid with vigorous stirring. Ice was added to the mixture to keep itchilled, the layers were separated and the aqueous layer was extractedwith ethyl acetate (600 ml). The ethyl acetate extract was combined withthe organic layer, and the solution was washed with acidified aqueoussaturated sodium chloride solution (300 ml, 2×). The solution was driedover magnesium sulfate, filtered, and the filtrate was divided into two735 ml portions.

To one 735 ml portion, sodium acetate (1.9 g) was added with stirring,and the resultant solution was allowed to stir for an additional 3 h atroom temperature. The precipitate was collected by suction filtrationand then washed with ethyl acetate. The resultant white crystals wereair-dried to yield 6.7 g of product, which, when dried in vacuo for 7 h,gave 6.1 g of sodium7-(S)-(2-phenoxyacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone: n.m.r. δ 3.94 (2xq, 4 total, C-2 and C-3' protons), 4.59 (s, 2,phenoxyacetamido methylene), 5.10 (d, 1, J^(C-7H) =2 Hz, C-6 proton),5.3 (dd, 1, J^(C-6H) =2Hz, J^(amide) =8.3 Hz, C-7 proton), 6.8-7.4 (m,5, aromatic), 9.4 (d, 1, J^(C-7H) =8.3 Hz, amido proton).

EXAMPLE 5 Benzhydryl 7-(S)-(2-phenoxyacetamido)-3hydroxymethyl-3-cephem-4-carboxylate sulfone

Effluent (1500 ml) from the IMER of Example 4 was layered with ethylacetate (1000 ml) and diphenyldiazomethane (12.0 g) was added to themixture. The mixture was chilled to 0° C. in an ice-alcohol bath, andthe pH of the mixture was adjusted with stirring to 2.5 by the additionof 1 N hydrochloric acid. After 0.33 h additional diphenyldiazomethane(1.2 g) was added to the solution. After 2 h the layers were separated,and the aqueous layer was extracted with ethyl acetate (400 ml, 2×). Theethyl acetate extracts were combined with the ethyl acetate layer, andthe resultant ethyl acetate solution was washed with 0.3 M phosphatesolution (2×, pH 7.0), then with aqueous saturated sodium chloridesolution (400 ml, 2×). The ethyl acetate solution was then dried overmagnesium sulfate, filtered and the solvent was removed in vacuo to givea syrup-like mass. A total of 1000 ml of methylene chloride was added tothe syrup, then removed in vacuo to yield 31.8 g of the benzhydryl esteryellow foam.

Part of the yellow foam (2.0 g) was triturated with diethyl ether (200ml), the triturate was filtered, and the filtrate evaporated toapproximately 1/3 of its original volume. The cream-colored solid whichprecipitated was collected by suction filtration and washed with ether,to yield 0.5 g of non-crystalline benzhydryl7-(S)-(2-phenoxyacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone: n.m.r. δ 4.07 (d, 2, C₃ '-H), 4.26 (ABq, 2, C₂ -H), 4.68 (s, 2,acetamido CH₂), 5.22 (dd, 1, C₇ -H), 5.26 (t, 1, hydroxy proton) 5.57(d, 1, C₆ -H), 6.96 (s, 1, benzhydryl methine), 7.2 to 7.6 (m, 15,aromatic protons), 9.22 (d, 1, amide proton).

EXAMPLE 6 Benzhydryl7-(S)-(2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylate sulfone

7-(S)-(2-Phenylacetamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid (15g) was dissolved in a buffer solution (0.05 M sodium citrate plus 0.075M sodium phosphate, pH 7.0, 1500 ml) while maintaining the pH of thesolution between pH 7.0-7.2 by the addition of 1 N sodium hydroxide. Thesolution was then percolated through the IMER (at 60 to 100 ml/h).

The collected effluent (1250 ml) was layered with ethyl acetate (600 ml)and the resultant emulsion was chilled to 0° C. in an ice-alcohol bath.Diphenyldiazomethane (5.0 g) was added to the stirred emulsion, and themixture was stirred for an additional 2.5 h, while the pH of thereaction mixture was maintained at about 2.5 by the addition of 1 Nhydrochloric acid.

The phases were separated and the aqueous phase was extracted with ethylacetate (400 ml, 2×). The ethyl acetate extracts and the ethyl acetatephase were combined, washed with 0.3 M sodium phosphate solution (pH 7,2×) and saturated aqueous sodium bicarbonate solution (300 ml, 1×). Thecombined ethyl acetate solution was dried over magnesium sulfate,filtered, and the solvent was removed in vacuo to give a yellow foam.The foam was pulverized, yielding 12.6 g of benzhydryl7-(S)-(2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone: n.m.r. δ 3.60 (s, 2, 2-phenylacetamido CH₂), 4.06 (s, 2, C₃'-H), 4.22 (ABq, 2, C₂ -H), 5.10 (dd, 1, C₇ -H), 5.47 (d, 1, C₆ -H),6.90 (s, 1, benzhydryl methine), 7.2 to 7.6 (m, 15, aromatic protons),9.07 (d, 1, amido proton).

EXAMPLE 7 Sodium7-(S)-(2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylate sulfone

7-(S)-(2-Phenylacetamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid(13.0 g) was dissolved in buffer (0.05 M sodium citrate plus 0.075 Msodium phosphate, pH 7.0, 1300 ml) while maintaining the pH of thesolution between pH 7.0-7.2 by the addition of 1 N sodium hydroxidesolution. This solution was percolated through the IMER (60 to 100ml/h).

The IMER was then washed with buffer. A portion of the collectedeffluent (1600 ml) was layered with ethyl acetate (1000 ml), and theemulsion was cooled to 0° in an ice-alcohol bath. The layers wereseparated, and the aqueous layer was extracted with ethyl acetate (300ml, 2×). The ethyl acetate extracts and the ethyl acetate layer werecombined, washed with acidified saturated sodium chloride solution (2×),dried over magnesium sulfate, and filtered. Sodium acetate (3.0 g) wasadded to the vigorously stirred filtrate, and the mixture was stirredfor an additional 4 h. The precipitate was collected by suctionfiltration, then dried in vacuo to give 9.75 g (78% yield) of sodium7-(S)-(2-phenylacetamido)-3-hydroxymethyl-3-cephem-4-carboxylatesulfone: F.A.B.M.S. (m+1)=403.

EXAMPLE 8 Sodium7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone

Sodium7-(S)-[2-(thien-2-yl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylatesulfone was dissolved in 0.3 M pH 7 phosphate buffer to yield a 2% w/vsolution which was then percolated through the IMER at 300 to 400 ml/hr.

A portion of the effluent from the above IMER procedure was chilled to0° in an ice-alcohol bath, layered with ethyl acetate, and the pH of theemulsion adjusted to 2.5 by the addition of 1 N hydrochloric acid.Immediately thereafter, the layers were separated, and the aqueous layerwas extracted with ethyl acetate. The ethyl acetate extract and theethyl acetate layer were combined, washed with aqueous saturated sodiumchloride solution, dried over magnesium sulfate, filtered and thefiltrate was evaporated to dryness in vacuo. The residue was dissolvedin methanol (1000 ml), and then a methanol solution of sodium acetate(110% of the equivalents required for neutralization of the 4-carboxylicacid) was added. The precipitate was collected by filtration, washedwith methanol and dried to yield 6.3 g of sodium7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone.

EXAMPLE 9 Benzhydryl7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone

To part of the effluent (1000 ml) from the IMER of Example 8 above wasadded ethyl acetate (500 ml) and the emulsion which formed was chilledto 0°. Diphenyldiazomethane (7.8 g) was added to the cold emulsion andthe pH was lowered to 2.5 by the addition of 1 N hydrochloric acid.After stirring for 1.5 h. the layers were separated, the aqueous layerextracted with ethyl acetate and the extract combined with the ethylacetate layer. The ethyl acetate solution was washed with acidifiedsaturated aqueous sodium chloride solution, dried over magnesium sulfateand filtered. To the filtrate were added a few seed crystals of theester and the solution was refrigerated for 4 days. The crystals werecollected by suction filtration, washed with cold ethyl acetate, thendried in vacuo to yield 13.2 g of cream-white crystals of benzhydryl7-(S)-[2-(thien-2-yl)acetamido]-3-hydroxymethyl-3-cephem-4-carboxylatesulfone: n.m.r. (Reference: tetramethylsilane) δ 3.85 (s, 2,2-(thien-2-yl) acetamido methylene protons), 4.02 (s, 2, C₃ '-H), 4.23(q, 2, C₂ -H), 5.11 (dd, 1, C₇ -H), 5.26 (t, 1, hydroxy proton) 5.50 (d,1, C₆ -H), 6.9 to 7.6 (m, 13, aromatic protons), 6.96 (s, 1, benzhydrylmethine proton), 9.12 (d, 1, amido proton).

EXAMPLE 10 Benzhydryl7-(S)-benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone

a. IMER preparation--A citrus acetylesterase IMER was prepared as aboveusing Fractosil 200 (23.7 g), 8% glutaraldehyde (100 ml) and citrusacetylesterase (31 ml). 2.5 h was allowed for esterase binding. Afterpacking the immobilized enzyme into the column, the column was washedwith buffer.

b.Deacylation--Sodium-7-(S)-benzyloxycarbamido-3-acetoxymethyl-3-cephem-4-carboxylatesulfone (17.45 g) was dissolved in 0.2 M phosphate solution (pH 7.0, 873ml) to yield a 2% solution of sulfone, and the pH of this solution wasadjusted from 6.6 to 6.9 with 1 N sodium hydroxide solution. Thesolution was then percolated through the column (40 to 50 ml/h) a totalof 3 times, followed by adding citrus acetylesterase (10 ml) directly tothe eluant. As the reaction mixture was percolating through the IMER, itwas necessary to periodically remove by filtration the precipitate thatformed in the solution on standing before it was percolated through theIMER.

Part of the eluant (610 ml) from the IMER was chilled to approximately3° C. in an ice-alcohol bath and an ethyl acetate solution (200 ml) ofdiphenyldiazomethane (3.25 g) was added. The pH of the emulsion wasadjusted to 2.6 with 1 N sulfuric acid. After the mixture was stirredfor 2 h. the layers were allowed to separate. The ethyl acetate layerwas washed with a 1:1 pH 7.0 buffer/sodium chloride solution (150 ml,4×), dried over magnesium sulfate, filtered, and the solvent wasevaporated in vacuo to give an orange-colored foam. The foam was dried(in vacuo) for 0.75 h. Chloroform (20 ml) was added to the orange foamfollowed by the addition of diethyl ether to facilitate precipitation ofthe product while stirring. Additional chloroform (5 ml) was added todissolve the gum that formed and sonification of this solution gavewhite crystals which were collected by filtration. Spectral analysisconfirmed that the crystals (5.45 g, 58%) were benzhydryl7-(S)-benzyloxycarbamido-3-hydroxymethyl-3-cephem-4-carboxylate sulfone:n.m.r. (100 MHz) δ 4.09 (d, 2, C₃ '-H), 4.25 (ABq, 2, C₂ -H), 5.04 (dd,1, C₇ -H), 5.26 (t, 1, hydroxy proton) 5.47 (d, 1, C₆ -H), 6.93 (s, 1,benzhydryl methine), 7.2 to 7.6 (m, 15, aromatic protons), 8.52 (d, 1,amido proton); analysis: calculated: C, 61.19: H, 4.59; N, 5.10; S,5.83; found: C, 61.78; H, 4.79; N, 5.41; S, 6.27.

EXAMPLE 11 Sodium7-(S)-[D-(5-(2,4-dichlorobenzoxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone

A 2% solution of7-(S)-[D-(5-(2,4-dichlorobenzoxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylic acid in0.2 M sodium citrate (pH adjusted to 7.0 with 1 N sodium hydroxide) waspercolated through the IMER (50 to 35 ml/h). Part of the eluant from theIMER (500 ml) was chilled in an ice-alcohol bath, and ice was added.Ethyl acetate (500 ml) was added to the cold eluant and the pH of themixture was adjusted to 2.5 by the addition of 1 N hydrochloric acid.The layers were separated, and the aqueous layer was extracted withethyl acetate (250 ml, 2×). The ethyl acetate layer and extracts werecombined, washed with saturated aqueous sodium chloride solution, driedover magnesium sulfate and filtered. Sodium acetate (3.12 g) was addedto the filtrate with stirring and stirring was continued for 4 h at roomtemperature. The crystals formed were collected by suction filtration,washed with ethyl acetate and air-dried to give 5.7 g of sodium7-(S)-[D-(5-(2,4-dichlorobenzoxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone:F.A.B.M.S. (m+1)=600.

EXAMPLE 12 Benzhydryl 7-(S)-[D-(5-(2,4-dichlorobenzoxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone

Benzhydryl 7-(S)-[D-(5-(2,4-dichlorobenzoxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-acetoxymethyl-3-cephem-4-carboxylate sulfone(14.7 g) was added to a 0.2 M sodium citrate solution (pH adjusted to7.0 by the addition of 1 N sodium hydroxide to yield a 1% solution ofsulfone), and the solution was percolated through an IMER (50 to 35ml/h). The eluant was refrigerated and diluted with ethyl acetate.Diphenyldiazomethane (10.0 g) was added to the cold eluant and the pH ofthe mixture was adjusted to 3.5 by the addition of 1 N hydrochloricacid. After stirring the mixture for 4 h, the layers were separated andthe aqueous layer was extracted with ethyl acetate (250 ml, 2×). Theethyl acetate layer and extracts were combined, washed with a saturatedsodium chloride solution, then dried over magnesium sulfate andfiltered. Diphenyldiazomethane (1.6 g) was added to the filtrate and thesolution was stirred overnight. Additional diphenyldiazomethane (1.0 g)was added. The mixture was stirred for approx. 6 h, and was thenevaporated to yield 22.3 g of yellow foam. The foam was triturated withdiethyl ether, the liquid decanted, the pale yellow solid dissolved inmethylene chloride, and the solution evaporated to dryness in vacuo. Thetrituration procedure was repeated yielding a foam (14.9 g, 78% yield)of benzhydryl7-(S)-[D-(5-(2,4-dichlorobenzoxycarbonylamino))-5-(benzhydrylcarboxylate)valeramido]-3-hydroxymethyl-3-cephem-4-carboxylate sulfone:n.m.r. (DMSO d₆) δ 1.40-2.45 (br. m., 6, valeramido methylenes), 4.04(d, 2, C₃ '-H), 4.11 (ABq, 2, C₂ -H), 4.54 (m, 1, valeramido methine),5.06 (dd, 1, C₇ -H, J=2.5, 8), 5.21 (t, 1, hydroxy proton), 5.40 (d, 1,C₆ -H, J=2.5), 6.79 (s, 1, benzhydryl methine on valeramido ester), 6.89(s, 1, benzhydryl methine on C-4 ester), 7.2 to 7.7 (br. m., 23,aromatic protons), 8.85 (d, 2, S-amido proton), 8.97 (d, 1, C-7 amidoproton).

I claim:
 1. A compound of the formula ##STR24## wherein R₁ is a. C₁ toC₇ alkyl, C₃ to C₇ alkenyl, chloromethyl, dichloromethyl,4-carboxybutyl, 4-formylbutyl, 4-protected carboxybutyl,4-amino-4-carboxybutyl or 4-protected amino-4-protected carboxybutyl;orb. C₁ to C₆ alkoxy, C₃ to C₆ cycloalkyloxy, benzyloxy or substitutedbenzyloxy, wherein the substituents are one to three groups chosen fromthe group consisting of C₁ to C₄ alkyl, C₁ to C₄ alkoxy and chloro; orc. 1,4-cyclohexadienyl, phenyl or substituted phenyl wherein thesubstituents are one or two groups chosen from the group consisting ofchlorine, bromine, hydroxy, trifluoromethyl, C₁ to C₄ alkyl, C₁ to C₄alkoxy, carboxy, carboxymethyl, hydroxymethyl, aminomethyl and protectedaminomethyl; or d. an arylalkyl group of the formula

    R'--(O).sub.m --CH.sub.2 --

wherein R' is 1,4-cyclohexadienyl, phenyl or substituted phenyl asdefined above, and m is zero or one; or e. a substituted arylalkyl groupof the formula ##STR25## wherein R" is R' as defined above, 2-thienyl,or 3-thienyl; W is hydroxy or protected hydroxy, carboxy or protectedcarboxy, amino or protected amino; or f. a heteroarylmethyl group of theformula

    R'"--CH.sub.2 --

wherein R'" is 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 2-thiazolyl,5-tetrazolyl or 1-tetrazolyl; andR₂ is hydrogen, a carboxylic acidprotecting group, lithium cation, sodium cation or potassium cation. 2.A compound of claim 1, wherein R₁ is C₁ to C₇ alkyl, C₃ to C₇ alkenyl,chloromethyl, dichloromethyl, 4-carboxybutyl, 4-formylbutyl, 4-protectedcarboxybutyl, 4-amino-4-carboxybutyl or 4-protected amino-4-protectedcarboxybutyl.
 3. A compound of claim 2, wherein R₁ is 4-protectedamino-4-protected carboxybut-1-yl.
 4. A compound of claim 3, wherein R₁is 4-(2,4-dichlorobenzyloxycarbonylamino)-4-(benzhydrylcarboxylate)but-1-yl.
 5. A compound of claim 4, wherein R₂ is hydrogen.6. A compound of claim 4, wherein R₂ is benzhydryl.
 7. A compound ofclaim 4, wherein R₂ is sodium cation.
 8. A compound of claim 1, whereinR₁ is C₁ to C₆ alkoxy, C₃ to C₆ cycloalkyloxy, benzyloxy or substitutedbenzyloxy, wherein the substituents are one to three groups chosen fromthe group consisting of C₁ to C₄ alkyl, C₁ to C₄ alkoxy and chloro.
 9. Acompound of claim 8, wherein R₁ is benzyloxy.
 10. A compound of claim 9,wherein R₂ is benzhydryl or sodium cation.
 11. A compound of claim 1,wherein R₁ is 1,4-cyclohexadienyl, phenyl or substituted phenyl.
 12. Acompound of claim 11, wherein R₁ is phenyl or p-methylphenyl.
 13. Acompound of claim 12, wherein R₂ is benzhydryl or sodium cation.
 14. Acompound of claim 1, wherein R₁ is an arylalkyl group of the formula

    R'--(O).sub.m --CH.sub.2 --.


15. A compound of claim 14, wherein R' is phenyl.
 16. A compound ofclaim 15, wherein R₂ is benzhydryl or sodium cation.
 17. A compound ofclaim 1, wherein R₁ is a heteroarylmethyl group of the formula

    R'"--CH.sub.2 --.


18. A compound of claim 17, wherein R'" is thien-2-yl.
 19. A compound ofclaim 18, wherein R₂ is benzhydryl or sodium cation.